The correlation between antimutagenic effect in Salmonella

Abstract Background: Antimutagenic effect of natural products such as plant extracts and honey bee is important in the discovery of new, effective cancer preventing agents. In this study, we aimed to determine the relationship between total phenolic content and antimutagenic activities of honey bee.
Material and methods: Total polyphenol content and antioxidant properties were determined by Folin–Ciocalteu colorimetric method, the total phenolic content was represented as mg gallic acid equivalents per g or ml of sample. Indeed, the antimutagenic effect was carried out against known mutagenic substances (sodium azide, 2-nitrofluorene, Mitomycine C, using Salmonella typhimurium TA98, TA100 strains for base pair substitution mutations and TA102 strain for frameshift mutation.
Results: the results obtained revealed that Algerian honey bee exhibited higher polyphenol content; it was between 121.44 and 204.52 µg gallic acid equivalent/mg of honey sample. In addition, all tested honey bee have shown an antimutagenic effect of positive mutagens with percentage of inhibition varied between 42.32% for honey sample 4 to 54.07% for honey sample 5. There is powerful correlation between total phenolic content and antimutagenic effect of honey bee (correlation coefficient =0.70).
Conclusion: the results obtained suggest that Algerian honey bee is effective as an antimutagenic agent; it can play an important role in the protection of mutagenic effect.
Keywords: AMES assay; antimutagenic effect; honey bee; Salmonella typhimurium; total polyphenolic content.

In recent years, there has been increasing interest in the identification of antimutagenic and anticarcinogenic components of human diet (Pariza, 1984; Graham, 1983; Ames, 1983). It is known that a variety of phenolic compounds are present in fruits and vegetables commonly consumed by humans. Several of these phenolic compounds have been shown to suppress carcinogen-induced mutagenesis and neoplasia in laboratory animals (Stich and Rosin, 1984; Wattenberg et al., 1980 ; Wood et al. 1982).
Honey has been used since ancient times as a flavorful sweetener and is considered a part of traditional folk medicine. It is a natural product consisting of a highly concentrated solution of a complex mixture of sugar and other constituents, such as minerals, proteins, vitamins, organic acids, flavonoids, phenolic acids, enzymes and volatile compounds. These contents present a wide range of biological effects such as antimicrobial, antioxidant and anti-inflammatory effects. However, the quality of honey bee depends largely on floral, geographical, seasonal and environmental factors and storage conditions.
Many mutagenic agents known as direct-acting mutagens, such as sodium azide (NaN3), directly affect genetic material that can causing point mutation in the genome and structural damage; the 4-nitro-o-phenylenediamine causes frame shift mutation. Mtomycin C; antibiotic used in therapy against many forms of human cancer, it can interact with biological molecules and can induce genetic hazards in non-tumor cells. One of the possible approaches to protect DNA from damage caused by these chemical mutagens and others mutagens is to use natural antimutagenic agents that are able to counteract the effects of mutagens. Hence, searching for antimutagenic compounds represents a rapidly expanding field of cancer research (Słoczyńska et al. 2014).
Due to their antimutagenic activities, phenolic compounds such as phenolic acids, naphthoquinones, xanthones, stilbenes, flavonoids, lignans, lignins and condensed tannins have become more important in the chemoprevention of cancer.
Algeria is an African country that occupies the centre of the continent; it is characterized by highly diversified forest ecosystems and a significant variation in climate that varies from the Mediterranean to the Sahara. These conditions offer a production of honey bees of good quality and very diversified. However, very few studies have been conducted on Algerian bees. Indeed, to our knowledge, this is the first study that reports the antimutagenic effect of the honey bee and its correlation with polyphenolic content. Therefore, we sought to determine the antimutagenic effect and the possible preventive capacity of processed honey bee from seven Algerian floral and geographic sources against the mutagenicity of x; y; and z and its relationship between polyphenolic content of honey bee.

Material and methods:
Honey sample
Floral source Geographic source
Honey Sample 1
Honey sample 2
Honey sample 3
Honey sample 4
Honey sample 5
Honey sample 6
Honey sample 7

Tester strains
Dosage of polyphenolic content
The total phenolic content was determined using the Folin- Ciocalteu method, and the results were expressed as µg gallic acid/mg of honey. Initially, honey solutions were prepared at a concentration of 0.1 g/mL. An aliquot of 0.5 mL of the stock solution was mixed with 0.3 mL of the Folin–Ciocalteu reagent and 2 mL of a 15% sodium carbonate solution. Distilled water was added to a final volume of 5 mL. Following incubation for 2 h, the absorbance of the reaction mixture was measured at 798 nm against a blank of distilled water. A standard curve of gallic acid was drawn within a concentration range of 7.0 x 10–4 to 7.8 x 10–3 mg/mL.
Antimutagenic effect
The antimutagenic effect of honey bee was achieved according to the assay described previously by Maron and Ames with small modifications. In brief, three strains of Salmonella typhimurium with three positive mutagens were used: 4-nitro-o-phenylenediamine for S. typhimurium 98, sodium azide for S. typhimurium 100 and mitomycin C for S. typhimurium 102. The presence of genetic markers has been systematically checked.
The bacteria were cultured in 20 ml of nutrient broth and incubated for 18 h at 37 °C with continuous agitation. 100 μl of overnight culture, 100 μl of each honey and 100 μl of positive mutagen are mixed with 500 μl phosphate buffer (13.8 g/L NaH2PO4 and 14.2g/L Na2HPO4). The mixture is pre-incubated at 37°C for 20 minutes.
A volume of 2 ml of molten soft agar medium with histidine and biotin solution was added aseptically to the mixture and poured after slight agitation onto a minimal glucose agar plate. After solidification of the mixture, the plates are incubated for 48 hours at 37°C. The number of revertant colonies on the plates was counted after incubation.
Honey is considered to have a mutagenic effect if the reversion coefficient (RC) is greater than or equal to 2. We calculated this coefficient according to this formula: RC= R1/R0
The antimutagenic effect is expressed as a percentage of inhibition (% of inhibition):
100 – (R3/R2X100)
Where: R0 is the number of spontaneous revertant colonies per plate
R1 is the number of revertant colonies induced by honey
R2 is the number of revertant colonies per plate exposed to positive mutagen
R3 is the number of revertant colonies per plate exposed to positive mutagen and honey.

Statistical analysis
Dosage of polyphenolic content

Antimutagenic effect
As shown in table X, none of the honey tested showed a mutagenic effect. However, all the honey samples have displayed significant antimutagenic effect in both base pair substitution (TA 98 and TA 100) and frameshift mutagenic assays (TA102).

The concentration and type of phenolic substances depend on the floral origin of the honey and are mainly responsible for its biological activities [1]. The total phenolic content (µg/mg of honey) was found to vary significantly (P=0..25), , which may be due to their different botanical and regional origins.
The results of the antimutagenic effect of honey have shown that all honey samples exhibited significant inhibition of positive mutagen; these results are the first that suggest the protective effect of honey bee against known mutagenic substances such as 4-nitro-o-phenylenediamine; sodium azide and mitomycin C. (Wang, Andrae, et Engeseth 2002)
Recent research has confirmed that many natural products contain components that can provide protection against chronic disease, including some forms of cancer. In this study, honey bee exhibit good antimutagenic activity against three mutagens and it can reduce mutant colonies in the S.typhimuruim TA98, TA100 and TA102 strains.

The antimutagenic effect of honey may be due to its antioxidant activity, presence of phenol and flavonoide
Also, to research molecular mechanism of tumor genesis and be able to offer biomedical preventive
benefits to tumor formation make these studies inevitable1.
showed an antimutagenic effect of quercitin against the mutagen 2AA. They also reported that flavonoids have been demonstrated to be effective inhibitors of mutagenicity induced in Salmonella by AFB1. The rates of mutagenic activity modulation observed in this study may indicate a contribution of flavonoids to the deactivation of AFB1, 2AF and, for some results, 2AA, or even a protective action of the M.ilicifolia infusion or its metabolites.

The results obtained in this study are very promotes: the is an reduction of mutagenic effect of the direct mutagens and the is strong correlation between antimutagenic effect of honey bee and its composition with total polyphenolic content.

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